Department of Ophthalmology, Shanghai General Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
J Diabetes Investig. 2018 Sep;9(5):1041-1051. doi: 10.1111/jdi.12815. Epub 2018 May 14.
AIMS/OBJECTIVE: The present study aimed to explore the effects of micro-ribonucleic acid-365 (miR-365) on apoptosis of retinal neurons by targeting insulin-like growth factor-1 (IGF-1) in diabetes mellitus rats.
High glucose-induced retinal neurons were assigned into the blank (with no plasmid transfection), negative control (with plasmid transfection), anti-miR-365 (transfected miR-365 antagomir), transfected IGF-1 short hairpin RNA plasmid (sh-IGF-1) and transfected miR-365 antagomir and IGF-1 shRNA plasmid (anti-miR-365 + sh-IGF-1) groups. Proliferation and apoptosis of retinal neurons were detected by 5-ethynyl-2'-deoxyuridine assay and Hoechst 33342 staining, respectively. Expressions of miR-365, IGF-1, Bcl-2-associated X protein (Bax) and Bcl-2 were determined by reverse transcription quantitative polymerase chain reaction and western blotting. A control group contained 10 healthy rats. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining was used to evaluate apoptosis of retinal neurons in rats.
In the anti-miR-365 group, the apoptosis rate and Bax expression were reduced in comparison with the negative control and blank groups, whereas the sh-IGF-1 and anti-miR-365 + sh-IGF-1 groups presented an opposite trend. Compared with the normal group, expressions of miR-365 and Bax were increased, and expressions of IGF-1 and Bcl-2 were decreased, with more apoptotic cells in diabetes mellitus rat models. The sh-IGF-1 group had lower Bax expression, and higher expressions of IGF-1 and Bcl-2 with fewer apoptotic cells. Additionally, Bax expression was upregulated, expressions of IGF-1 and Bcl-2 were downregulated, and apoptotic cells were higher in the anti-miR-365 + sh-IGF-1 groups than the anti-miR-365 group.
The results of the present study suggest that suppressed miR-365 increases the IGF-1 expression, leading to anti-apoptotic effects on retinal neurons in diabetic rats.
本研究旨在探讨微小 RNA-365（miR-365）通过靶向胰岛素样生长因子-1（IGF-1）对糖尿病大鼠视网膜神经元凋亡的影响。
高糖诱导的视网膜神经元被分为空白组（无质粒转染）、阴性对照组（质粒转染）、抗 miR-365 组（转染 miR-365 拮抗剂）、转染 IGF-1 短发夹 RNA 质粒组（sh-IGF-1）和转染 miR-365 拮抗剂和 IGF-1 shRNA 质粒组（anti-miR-365+sh-IGF-1）。通过 5-乙炔基-2'-脱氧尿苷检测和 Hoechst 33342 染色分别检测视网膜神经元的增殖和凋亡。采用逆转录定量聚合酶链反应和蛋白质印迹法检测 miR-365、IGF-1、Bcl-2 相关 X 蛋白（Bax）和 Bcl-2 的表达。对照组包含 10 只健康大鼠。末端脱氧核苷酸转移酶 dUTP 缺口末端标记染色用于评估大鼠视网膜神经元的凋亡。
在抗 miR-365 组中，与阴性对照组和空白组相比，细胞凋亡率和 Bax 表达降低，而 sh-IGF-1 和 anti-miR-365+sh-IGF-1 组则呈现相反的趋势。与正常组相比，糖尿病大鼠模型中 miR-365 和 Bax 的表达增加，IGF-1 和 Bcl-2 的表达减少，凋亡细胞增多。sh-IGF-1 组 Bax 表达降低，IGF-1 和 Bcl-2 表达升高，凋亡细胞减少。此外，anti-miR-365+sh-IGF-1 组 Bax 表达上调，IGF-1 和 Bcl-2 表达下调，凋亡细胞较 anti-miR-365 组增多。
本研究结果表明，抑制 miR-365 可增加 IGF-1 的表达，从而对糖尿病大鼠视网膜神经元产生抗凋亡作用。
